Selenium is an essential component of the human diet because it is incorporated into at least 25 human proteins whose functions are required for key elements in human health, including the regulation of inflammation, male fertility and cellular antioxidant activity. Although it is known that selenium is incorporated in the form of the 21st amino acid, selenocysteine (Sec), the mechanism by which Sec incorporation occurs is unknown. To date, four factors are known to be required for the conversion of a UGA stop codon into one encoding Sec in eukaryotes: a cis-sequence in the selenoprotein mRNA termed a Sec insertion sequence (SECIS) element, the Sec-specific elongation factor (eEFSec) that carries the Sec-tRNASec, and a SECIS binding protein, SBP2. While these four factors are essential for Sec incorporation, their mechanism of action has not been determined. The overall goal of this work is to decipher the mechanism by which Sec incorporation is achieved with a focus on elongation factor specificity as well as the identification of novel factors that promote efficient and processive Sec incorporation. Our guiding hypothesis is that an SBP2/SECIS complex provides a signal to the eEFSec ternary complex (eEFSec/Sec-tRNASec/GTP) that allows it to bind the ribosomal A site, and that this mechanism is modified by as-yet unidentified factors to yield an efficient and processive reaction. To test this hypothesis, three specific aims are proposed. First, we will identify the determinants for specificity in the Sec-specific elongation factor, eEFSec. Since eEFSec functions analogously and in parallel with canonical translation elongation, we propose to analyze eEFSec function in the context of the known functions of the canonical homolog eEF1A, including the development of a novel eEFSec assay system and a determination of the role GTP hydrolysis in Sec incorporation. Second, we will determine the mechanism of eEFSec function and regulation. The recruitment of eEFSec by the SBP2/SECIS complex is likely an integral part of the Sec incorporation reaction, thus we propose to elucidate the mechanism, conformational consequences and amino acid sequences required for SBP2/ SECIS/eEFSec complex formation. Finally, we propose the identification and characterization of the novel factors required for processive and efficient Sec incorporation. SBP2 and eEFSec may be sufficient for basal Sec incorporation, but these two factors are unable to support efficient Sec incorporation in vitro or in transfected cells. Since both efficiency and processivity are essential features of selenoprotein production in vivo, we propose to identify the factors and cis-elements required, thus expanding the field to include specific regulators of the basal Sec incorporation machinery. Together these three aims represent an integrated molecular approach to identifying the mechanism of Sec incorporation in order to fulfill the long term goal of enhancing selenoprotein function in vivo.